The present invention relates generally to isolated and purified proteins that modulate SHP-2 biological activity and modulate cell signaling, and to nucleic acids encoding the same. More particularly, the present invention relates to an isolated and purified transmembrane protein designated as xe2x80x9cprotein zero relatedxe2x80x9d or xe2x80x9cPZRxe2x80x9d that binds the tyrosine phosphatase SHP-2, and an isolated and purified polynucleic acid encoding the same.
Protein tyrosine phosphatases (PTPs) represent a highly diverse family of enzymes that have a pivotal role in cell proliferation, differentiation, and transformation. Fischer, E. H., Charbonneau, H., and Tonks, N. K. (1991) Science 253:401-6; Walton, K. M. and Dixon, J. E. (1993) Annu. Rev. Biochem. 62:101-20; Hunter, T. (1995) Cell 80:225-236. SHP-1 and SHP-2, representing a subfamily of PTPs containing SH2 domains have been extensively studied in recent years. Zhao, Z, Shen, S. H. and Fischer, E. H. (1995) Adv. in Protein Phosphatases 9:297-317; Streuli, M. (1996) Curr. Opinion in Cell Biol. 183: 182-188; Scharenberg, A. M. and Kinet, J. P. (1996) Cell 87:961-964; Tonks, N. K., and Neel, B. G. (1996) Cell 87:365-368; Frearson, J. A. and Alexander, D. R. (1997) Bioessays 19;417427; Ulyanova, T., Blasioli, J., and Thomas, M. L. (1997) Immunolog. Res. 16:101-113; Byon, J. C., et al. (1997) Proc. Soc. Exp. Biol. and Med. 216:1-20; Neel, B. G. and Tonks, N. K. (1997) Curr. Opin. Cell. Biol. 9:193-204.
SHP-1 and SHP-2 share nearly 60% overall sequence identity and are regulated in similar manners. Nevertheless, in many systems, they have distinct physiological functions. SHP-1 has a negative role in proliferation of hematopoietic cells whereas SHP-2 is a positive transducer of growth factor signal transduction. This distinction in functions is presumably due to different physiological targets.
Recently, a number of putative substrates of SHP-1 and SHP-2 have been identified. Xiao, S., et al. (1994) J. Biol. Chem. 269:21244-21248; Milarski, K. L. and Saltiel, A. L. (1994) J. Biol. Chem. 269:21239-21243; Noguchi, T., et al. (1994) Mol. Cell. Biol. 14:6674-6682; Yamauchi, K., et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92:664-668; Yamauchi, K., et al. (1995) J. Biol. Chem. 270:17716-17722; Frearson, J. A., Yi, T., and Alexander, D. R. (1996) Eur. J. Immunol. 26:1539-1543; Valiante, N. M., et al. (1996) J. Exp. Med. 184:2243-2250; Carlberg, K. and Rohrschneider, L. R. (1997) J. Biol. Chem. 272:15943-15950; Ruff, S. J., Chen, K., and Cohen S. (1997) J. Biol. Chem. 272:1263-1267; Gu, H., Griffin, J. D., and Neel, B. G. (1997) J. Biol. Chem. 272:16421-16430; Jiao, H., et al. (1997) Exp. Hematol. 25:592-600. One of them, designated as SIRP or SHPS-1, has been cloned (Kharitonenkov, A., et al. (1997) Nature 386:181-186; Fujioka, Y., et al. (1996) Mol. Cell. Biol. 16:6887-6899). Overexpression of catalytically inactive mutants of SHP-1 and SHP-2 resulted in the identification of several hyper-phosphorylated proteins associated with the inactive SHP-1 and/or SHP-2 (Zhao, Z., et al. (1995) J. Biol. Chem. 270:11765-17769; Su, L., et al. (1996) J. Biol. Chem. 271:10385-10390.
Although a number of putative substrates of SHP-2 have been identified, little is known at the molecular level about the signaling mechanisms of SHP-2. This lack of knowledge represents a serious deficiency in the art in view of the effects of SHP-2 as described above. Therefore, further characterization of SHP-2 signaling in vertebrates, particularly in mammals, and more particularly in humans is needed. A novel isolated and purified polypeptide having a role in SHP-2 signaling would have broad utility in view of the above-described various and multiple physiological roles of SHP-2.
The present invention contemplates an isolated and purified vertebrate protein, referred to herein as xe2x80x9cprotein zero relatedxe2x80x9d or xe2x80x9cPZRxe2x80x9d, which plays a role in SHP-2-mediated signaling. More preferably, a polypeptide of the invention is a recombinant polypeptide. Even more preferably, a polypeptide of the present invention comprises a mammalian PZR. Even more preferably, a polypeptide of the present invention comprises a human PZR. Even more preferably, a polypeptide of the present invention comprises the amino acid residue sequence of any of SEQ ID NOs:1-8 and 17-48.
The present invention also provides an isolated and purified polynucleotide that encodes a polypeptide that plays a role in SHP-2-mediated signaling. In a preferred embodiment, a polynucleotide of the present invention comprises a DNA molecule from a vertebrate species. A preferred vertebrate is a mammal. A preferred mammal is a human. More preferably, a polynucleotide of the present invention encodes a polypeptide designated PZR. Even more preferred, a polynucleotide of the present invention encodes a polypeptide comprising the amino acid residue sequence of any of SEQ ID NOs:1-8 and 17-48. Most preferably, an isolated and purified polynucleotide of the invention comprises the nucleotide base sequence of any of SEQ ID NOs:1-8 and 17-48.
In another embodiment, the present invention provides an antibody immunoreactive with a PZR polypeptide as described above. SEQ ID NOs:1-8 and 17-48 sets forth nucleotide and amino acid sequences from representative vertebrates, human and mouse. Also contemplated by the present invention are antibodies immunoreactive with homologues or biologically equivalent PZR polynucleotides and polypeptides found in other vertebrates. Preferably, an antibody of the invention is a monoclonal antibody. More preferably, the PZR polypeptide comprises human PZR. Even more preferably, the PZR polypeptide comprises the amino acid residue sequence of any of SEQ ID NOs:1-8 and 17-48.
In another aspect, the present invention contemplates a process of producing an antibody immunoreactive with a PZR as described above, the process comprising: (a) transfecting a recombinant host cell with a polynucleotide that encodes a PZR polypeptide having a SHP-2 activity-modulating function; (b) culturing the host cell under conditions sufficient for expression of the polypeptide; (c) recovering the polypeptide; and (d) preparing the antibody to the polypeptide. SEQ ID NOs:1-8 and 17-48 set forth nucleotide and amino acid sequences from representative vertebrates, human and mouse. Preferably, the host cell is transfected with the polynucleotide of any of SEQ ID NOs:1-8 and 17-48. Even more preferably, the present invention provides an antibody prepared according to the process described above. Also contemplated by the present invention is the use of homologues or biologically equivalent polynucleotides and polypeptides found in other vertebrates to produce antibodies.
Alternatively, the present invention provides a process of detecting a PZR polypeptide as described above, wherein the process comprises immunoreacting the polypeptide with an antibody prepared according to the process described above to form an antibody-polypeptide conjugate, and detecting the conjugate.
In yet another embodiment, the present invention contemplates a process of detecting a messenger RNA transcript that encodes a PZR polypeptide as described above, wherein the process comprises hybridizing the messenger RNA transcript with a polynucleotide sequence that encodes that polypeptide to form a duplex; and detecting the duplex. Alternatively, the present invention provides a process of detecting a DNA molecule that encodes a PZR polypeptide as described above, wherein the process comprises hybridizing DNA molecules with a polynucleotide that encodes a PZR polypeptide having a SHP-2 binding function to form a duplex; and detecting the duplex.
In another aspect, the present invention contemplates an assay kit for detecting the presence of a PZR polypeptide in a biological sample, where the kit comprises a first container containing a first antibody capable of immunoreacting with a vertebrate PZR polypeptide having a SHP-2 binding function, with the first antibody present in an amount sufficient to perform at least one assay. Preferably, an assay kit of the invention further comprises a second container containing a second antibody that immunoreacts with the first antibody. More preferably, the antibodies used in an assay kit of the present invention are monoclonal antibodies. Even more preferably, the first antibody is affixed to a solid support. More preferably still, the first and second antibodies comprise an indicator, and, preferably, the indicator is a radioactive label or an enzyme.
In an alternative aspect, the present invention provides an assay kit for detecting the presence, in biological samples, of a PZR polypeptide, the kits comprising a first container that contains a second polynucleotide identical or complementary to a segment of at least 10 contiguous nucleotide bases of a polynucleotide that encodes a PZR polypeptide having a SHP-2 binding function.
In another embodiment, the present invention contemplates a diagnostic assay kit for detecting the presence, in a biological sample, of an antibody immunoreactive with a PZR polypeptide, the kit comprising a first container containing a PZR polypeptide having a SHP-2 binding function that immunoreacts with the antibody, with the polypeptide present in an amount sufficient to perform at least one assay.
In still a further embodiment, this invention pertains to therapeutic methods based upon the SHP-2 binding function of PZR as described herein. Such therapeutic methods include administration of a soluble form of the PZR protein and gene therapy approaches using an isolated and purified polynucleotide of the present invention. Therapeutic methods in accordance with the present invention are also contemplated to have application in the treatment of type 1B Charcot-Marie-Tooth disease.
Thus, a key aspect of this invention pertains to the discovery of the novel PZR protein and nucleic acid encoding the PZR protein. Preferred nucleic acid and amino acid sequences for PZR are described in any of SEQ ID NOs:1-8 and 17-48.
It is another aspect of this invention that the novel PZR protein binds with SHP-2 to modulate SHP-2 biological activity.
It is thus another aspect of this invention to provide a purified and isolated PZR polypeptide having a SHP-2 binding function.
The foregoing aspects and embodiments have broad utility given the biological significance of SHP-2, as is known in the art. By way of example, the foregoing aspects and embodiments are useful in the preparation of screening assays and assay kits that are used to identify compounds that affect or modulate PZR or SHP-2 biological activity, or that are used to detect the presence of the proteins and nucleic acids of this invention in biological samples. Additionally, it is well known that isolated and purified polypeptides have utility as feed additives for livestock and further polynucleotides encoding the polypeptides are thus useful in producing the polypeptides.
Some of the aspects and objects of the invention having been stated hereinabove, other aspects and objects will become evident as the description proceeds, when taken in connection with the accompanying Examples and Drawings as best described hereinbelow.